DESCRIPTION: (Applicant's Abstract) Opioid analgesic drugs, including heroin and morphine, form a major class of drugs with potential for abuse. Different opioid agonists exhibit different efficacies in various biological systems, and agonist efficacy may not only determine the response produced by the drugs, but also contribute to the development of tolerance to and dependence upon the drug. The proposed project will examine the signal transduction mechanisms underlying agonist efficacy and intrinsic efficacy at mu and delta opioid receptors in two well-established cell culture model systems: mMOR-CHO cells that have been transfected with the mouse mu opioid receptor, SK-N-SH neuroblastoma cells, which contain primarily, mu receptors, and NG108-15 neuroblastoma x glioma cells which contain only delta opioid receptors. Agonist-stimulated [35S]-GTPgS binding, and agonist displacement of [3H]antagonist binding, will be used to measure agonist efficacy and intrinsic efficacy. Efficacy will be defined as maximal stimulation of [35S]GTPgS binding and intrinsic efficacy as both the maximal stimulation and the ratio of Ki in competition binding assays to ED50 in [35S]GTPgS assays. Scatchard analysis of agonist-stimulated [35S]GTPgS binding will be determined whether agonists of different efficacies change the affinity or number of activated G-proteins. Catalytic amplification factors will be calculated by comparing the receptor Bmax to the agonist-stimulated GTPgS binding Bmax, and the role of GDP and sodium in regulating agonist efficacy will be examined. The second phase of the project will examine the relationship between drug efficacy and receptor desensitization and downregulation after chronic drug treatment of cells. In these experiments, cells will be chronically treated with agonists of different efficacies to determine how much the response is decreased, to what extent cross-desensitization to the response produced by drugs of different efficacies develops, and the signal transduction mechanisms underlying the loss in agonist efficacy after chronic drug treatment. In the last phase of the proposed project, the contribution of receptor:transducer ratio and specific transducer reserve to agonist efficacy will be addressed using irreversible antagonists, pertussis toxin, and molecular transfection. A cDNA encoding a cloned Gia2 subunit will be transfected into cells to determine the contribution of specific transducer reserve to opioid agonist efficacy. Thus, the studies in this project are aimed at a mechanistic understanding of how opioid agonist efficacy is produced, and how desensitization effects agonist efficacy, at the level of the receptor-transducer interaction.